Scarab Genomics has established a unique, tiny footprint, extended bacterial fermentation process enabled by its reduced genome E. coli, for production of protein therapeutics, especially vaccine conjugates. The process will massively reduce the cost and upgrade the quality of these critical vaccine components. This Phase IIB resubmission focuses on commercial development of Scarab?s first vaccine product, the diphtheria mutant toxin CRM197, an important component of highly successful conjugate vaccines, of significant interest to the mission of the NIH. CRM197 has also shown promise as an anti-cancer agent. In high demand worldwide, it is difficult to produce by batch technology, so supply is constrained and unreliable. For Scarab to enter the vaccine market, cGMP-grade product is required. For maximum efficiency, Scarab proposes to outsource initial production of cGMP-grade CRM197 to a company that will use Scarab?s innovative process in its GMP facility, validating the process for GMP manufacturing. In a previous Phase 2 SBIR project, our process generated high levels of pure recombinant CRM197 and yielded consistently high quality soluble and stable CRM197 protein. In particular, the A and B breakdown products are minor components of Scarab?s high-quality product. The downstream process (DSP) from Phase 2 resulted in highly pure CRM197 now sold on the research market. Since the original Ph2B proposal, new research has established (i) successful scale up to a 10L productive fermentation process and (ii) a dramatically more modern and efficient DSP that is automatable, a vital requirement for commercial viability. Waisman Biomanufacturing (WB), a well-established not-for-profit cGMP contract development and manufacturing organization, will perform production of cGMP-grade CRM197. WB was selected because it has provided a very competitive quote, and has exceptional experience and expertise ? it has manufactured (and its clients have released) well over 300 clinical-grade products, with 5-10 investigational new drug applications submitted by their partners each year. WB?s location in the same city as Scarab will facilitate technology transfer and project oversight. The Specific Aims are: 1) Full development, scale-up and automation of the new DSP. Late-stage development of the fermentation process at Scarab, including four pilot fermentations and downstream processing, technology transfer to WB, and cGMP process development. 2) engineering GMP prototype run, specifications regarding purity and potency established. 3) Three GMP production runs that formulize within-run and post-purification specifications. 4) Final product validation that will include purity and potency assays (conjugation and human immunogenicity). Stability studies by Nitto Avecia. Materials from GMP engineering and production runs will provide potential customers with samples for evaluation. Project information will be used in a Biological Master File at the FDA.